Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. Hypoxia/contraction in muscle also stimulates glucose transport activity and this effect is additive to that of insulin. However, quantification of these processes by classical subcellular fractionation techniques has been very difficult because of the contaminating microfibrillar protein and dynamic studies at the molecular level have been almost impossible. Thus, we have prepared a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. Western blotting and confocal microscopy confirm the specificity and levels of HA-GLUT4-GFP expression in intact muscles, and isolated FDB muscle fibers and cardiomyocytes (Fazakerley et al.). Confocal microscopy further demonstrates that HA-GLUT4-GFP is translocated to PM and the t-tubules of isolated FDB fibers to the same degree after insulin and hypoxia, and that these effects are additive. However, TIRFM reveals a remarkable difference in dynamics between adipose and muscle cells: In muscle, in either isolated FDB fibers or soleus muscles in vitro, or in gastrocnemius muscles in living animals, in the non-stimulated state almost all GLUT4 are found in PM clusters and only very few are mobile. Furthermore, stimulation of 2-DOG transport in muscle by insulin is typically 2-3-fold, the same magnitude as that of the stimulation by insulin of fusion events at the GLUT4 clusters in adipose cells (2-fold, Stenkula et al.). Finally, the Ploug laboratory (Lauritzen et al.) has reported that in skeletal muscle, insulin reversibly stimulates local depletion of GLUT4 storage vesicles at sarcolemma and t-tubules rather than inducing movement of intact storage vesicles. These data together strongly suggest that GLUT4 clusters (called hubs in adipose cells) are the major site of glucose transport regulation in muscle, and that GLUT4 are released from these hubs in response to stimuli, and recycle through these hubs during, and reaccumulate in these hubs after, stimulation.